Comparison of mechanical and electrical activity and interstitial cells of Cajal in urinary bladders from wild-type and W/Wv mice

Abstract

Background and purpose: W/W(v) and wild-type murine bladders were studied to determine whether the W/W(v) phenotype, which causes a reduction in, but not abolition of, tyrosine kinase activity, is a useful tool to study the function of bladder interstitial cells of Cajal (ICC).

Experimental approach: Immunohistochemistry, tension recordings and microelectrode recordings of membrane potential were performed on wild-type and mutant bladders.

Key results: Wild-type and W/W(v) detrusors contained c-Kit- and vimentin-immunopositive cells in comparable quantities, distribution and morphology. Electrical field stimulation evoked tetrodotoxin-sensitive contractions in wild-type and W/W(v) detrusor strips. Atropine reduced wild-type responses by 50% whereas a 25% reduction occurred in W/W(v) strips. The atropine-insensitive component was blocked by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both tissue types. Wild-type and W/W(v) detrusors had similar resting membrane potentials of -48 mV. Spontaneous electrical activity in both tissue types comprised action potentials and unitary potentials. Action potentials were nifedipine-sensitive whereas unitary potentials were not. Excitatory junction potentials were evoked by single pulses in both tissues. These were reduced by atropine in wild-type tissues but not in W/W(v) preparations. The atropine-insensitive component was abolished by pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid in both preparations.

Conclusions and implications: Bladders from W/W(v) mice contain c-Kit- and vimentin-immunopositive ICC. There are similarities in the electrical and contractile properties of W/W(v) and wild-type detrusors. However, significant differences were found in the pharmacology of the responses to neurogenic stimulation with an apparent up-regulation of the purinergic component. These findings indicate that the W/W(v) strain may not be the best model to study ICC function in the bladder.

Figure 1

c-Kit-positive cells in wild-type and W/W^v bladder. c-Kit-positive cells in whole-mount preparations of wild-type (a,b) and W/W^v (d,e) mouse detrusor. Immunoreactive cells were elongated with several small lateral branches and were orientated parallel to the detrusor muscle fibres. Double-labelling with an antibody to vesicular acetylcholine transferase (vAChT, green) and anti-c-Kit (red) revealed close proximity between interstitial cells of Cajal and cholinergic neurons in both tissue types (c,f). All images are projections of a stack of optical sections captured with a confocal microscope.

Figure 2

Vimentin-positive cells in wild-type and W/W^v bladder. Vimentin-positive cells in whole-mount preparations of wild-type (a,b) and W/W^v (c,d) mouse detrusor. Vimentin (intermediate) filaments are found in interstitial cells of Cajal (ICC) and fibroblasts but not in smooth muscle cells and vimentin antibodies are often used to label ICC. Vimentin-positive cells had several morphologies including rounded, stellate or elongated cell bodies with branched processes and are likely to include both ICC and fibroblasts. All images are projections of a stack of optical sections captured with a confocal microscope.

Figure 3

Contractile responses to electrical field stimulation. Detrusor strips from wild-type (a) and W/W^v (b) animals contracted in response to electrical field stimulation applied for 10 s at frequencies of 1, 2, 4, 8 and 16 Hz; pulse width durations of 0.3 ms. All contractions were abolished in the presence of tetrodotoxin (TTX) (0.5 µmol·L⁻¹).

Figure 4

Cholinergic and purinergic components of neurogenic-evoked contractions. Detrusor strips from wild-type (a) and W/W^v (b) animals contracted in response to electrical field stimulation as before. In both tissue types, atropine (1 µmol·L⁻¹) reduced the amplitude of the contractions with the remaining responses inhibited by pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (30 µmol·L⁻¹). Summary graphs (c) reveal that atropine blocked approximately 50% of the contraction in wild-type strips, whereas in W/W^v strips, atropine reduced the contractions by only about 25%. Contraction amplitude at 16 Hz in wild-type strips was 52% cholinergic and 48% purinergic (control 571 ± 185 mg reduced to 272 ± 97 mg in atropine and further reduced to 24 ± 8 mg by PPADS), whereas responses in W/W^v strips were comprised of 27% cholinergic and 73% purinergic components (control 1054 ± 115 mg, atropine 774 ± 115 mg and PPADS 76 ± 15 mg).

Figure 5

Exogenously applied carbachol. Carbachol was applied to detrusor strips from wild-type (a) and W/W^v (b) bladders as indicated on the traces. Agonist was applied for 2 min and tissues were subsequently washed for at least 20 min before the next concentration was added.

Figure 6

Intracellular recordings of spontaneous electrical activity. Wild-type and W/W^v detrusor preparations exhibited electrical activity comprising spontaneous unitary potentials (denoted by asterisks) and larger events resembling action potentials (indicated by arrowheads). Nifedipine (1 µmol·L⁻¹) inhibited action potentials in both tissue types; however, unitary potentials were unaffected (trace not shown).

Figure 7

Neurogenic-evoked electrical responses. Wild-type and W/W^v detrusor preparations were stimulated with single pulses having durations of 0.1, 0.3 and 0.5 ms, which typically evoked excitatory junction potentials (EJPs). In the absence of drugs, similar responses were elicited in both wild-type and W/W^v tissues. Application of atropine (1 µmol·L⁻¹) caused a small reduction in the amplitude of the EJPs in wild-type preparations but did not inhibit those from W/W^v detrusors (see expanded traces of the responses to a pulse of 0.3 ms). In contrast, PPADS (100 µmol·L⁻¹) abolished the atropine-resistant component of EJPs in all tissues.